Background: There is no certified control material for hemoglobin analysis which has the hemoglobin (Hb)A 2 /E level as high as found in β-thalassemia trait, HbE trait, β-thalassemia/HbE disease and homozygote of HbE, the thalassemia types found frequently in the Southeast Asian population. The aim of this study was to prepare the lyophilized hemoglobin control materials for hemoglobin analysis. Methods: Washed and dialysed erythrocytes of normal individuals and patients with β-thalassemia trait, HbE trait, β-thalassemia/HbE disease, homozygous HbE were lysed in 5 sucrose solution. The lyophilized hemoglobin control materials were prepared by using a freeze-drying (lyophilization) method. The high performance liquid chromatography (HPLC) analysis of lyophilized hemoglobin was performed after storing at 20°C for 1, 15 and 30days and for 3months. Results: The chromatograms of lyophilized hemoglobin control materials showed similar patterns and similar levels of HbA, HbA 2 /E and HbF when compared with equivalent fresh whole blood. Moreover, the lyophilized hemoglobin presented a good correlation coefficient (r > 0.990) of relationships between HPLC, low pressure liquid chromatography (LPLC) and capillary electrophoresis (CE) methods. Conclusions: The lyophilized hemoglobin could be developed and used as control materials for hemoglobin analysis. © 2012 by Walter de Gruyter Berlin Boston.